Novel cd3 epsilon immunogens and antibodies

ABSTRACT

The present invention relates to novel CD3 epsilon peptides, antibodies against the novel CD3 epsilon peptides. The invention also relates to methods of identifying an immunodeficiency (such as severe combined immunodeficiency (SCID) or a T cell immunodeficiency) in a patient, which may involve antibodies against CD3 epsilon peptides.

INCORPORATION BY REFERENCE

This application claims priority to U.S. provisional patent applicationSerial Nos. 61/310,199 filed Mar. 3, 2010, 61/377,833 filed Aug. 27,2010 and 61/434,745 filed Jan. 20, 2011.

FEDERAL FUNDING LEGEND

This invention was supported, in part, by NIH Contract ADB-NO1-DK-6-3430(HHSN267200603430, K Pass PI), Novel Technologies in Newborn Screening,and, Luminex, Corp. This application was also supported in part by CDCCooperative Agreement (1U01EH000362, A. Comeau PI), Implementing SCIDNBS with Multiplexed Assays in an Integrated Program Approach. Thefederal government may have certain rights to this invention.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a novel CD3 epsilon peptides,antibodies against the CD3 epsilon peptides and diagnostic kits fordetecting CD3 epsilon peptides.

BACKGROUND OF THE INVENTION

SCID (severe combined immunodeficiency) presents one of the greatestopportunities for newborn screening (NBS), and also one of its mostdifficult challenges. This is the first condition detectable by NBS forwhich a cure is available, when identified and treated in early infancy(Railey et al., J Peds 2009; 155(6):834-40). However, the only testcurrently available with the potential of detecting SCID in the Guthriespecimen is the TREC (T-cell recombinant excision circle) assay (Chan &Puck, J Allergy Clin Immunol 2005; 115:391-8). The TREC assay presentstechnical challenges in that it is a primary screening assay using DNA,a protocol not accepted universally by the screening community (Green &Pass, Nat Rev Genet 2005; 6:147-55). Alternatively, immunoassays areused routinely in NBS as a first-tier screening protocol (Moyer et al.,Hastings Cent Rep 2008; 38(3):32-39).

SCID (severe combined immunodeficiency) fulfills the requirements for anewborn screening (NBS) condition: sufficient prevalence (estimated at1:50000 to 1:100000); biomarkers in the Guthrie specimen; and theavailability of an appropriate therapy (bone marrow transplant). Anassay to identify SCID and other T-cell deficiencies throughquantification of T-cell recombinant excision circles (TRECs) hasalready been developed and is currently being used in pilot studies inWisconsin and Massachusetts. In those two states, nearly 200,000newborns have been screened without a SCID baby having been found, butother T-cell immunodeficiencies were identified in the screening.Because the TREC assay utilizes DNA technology, those laboratories withlittle experience, equipment or personnel in molecular biology may facechallenges not encountered with an immunoassay. There exists a need foran alternative test for identifying alternate T-cell immunodeficienciesas well as alternate diagnostic kits.

SCID may result from a severe defect in both the T & B lymphocytesystems that leads to their diminishing. The absence of T-cells may be adefining characteristic of SCID and of other T-cell immunodeficiencies.(Edgar, J Clin Pathol 2008; 61:988-93). Because CD3 is part of theT-cell receptor complex on mature T-cells, it can be used as a markerfor deficiency of T-cells (Dava, Immunol Rev. 2009; 232:22-33). Two casereports of CD3 deficiency causing immunodeficiency have been reported(Roberts et al., Blood 2007; 109:3198-3206, Rieux-Laucat et al., N EnglJ Med. 206; 354:1913-21). CD45, a common antigen present on alldifferentiated lymphocytes, provides an internal control for the assay.(Eley, Curr Allergy and Clin Immunol. March 2008:21:1-24).

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention relates to, in part, Applicants' development ofnovel CD3 epsilon peptides, antibodies against the novel CD3 epsilonpeptides. The antibodies of the present invention may be used toidentify a CD3 deficiency in newborns.

In one embodiment, the invention pertains to an isolated CD3 epsilonpeptide, in particular CD3 epsilon peptides which may comprise, consistessentially of or consist of residues 8-12 of SEQ ID NO: 1 or residues6-10 of SEQ ID NO: 2. The invention also pertains to isolated antibodiesagainst CD3 epsilon peptides, in particular CD3 epsilon peptides whichmay comprise, consist essentially of or consist of residues 8-12 of SEQID NO: 1 or residues 6-10 of SEQ ID NO: 2.

In another embodiment, the invention relates to a method of identifyingan immunodeficiency in a patient, comprising isolating a blood samplefrom the patient and performing an immunoassay on the blood sample withan antibody against CD3 epsilon peptides, in particular CD3 epsilonpeptides which may comprise, consist essentially of or consist ofresidues 8-12 of SEQ ID NO: 1 or residues 6-10 of SEQ ID NO: 2. In anadvantageous embodiment, the immunodeficiency is severe combinedimmunodeficiency (SCID) or a T cell immunodeficiency.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIG. 1 depicts the amino acids CD3 epsilon peptides SEQ ID NOS: 1 and 2;

FIG. 2 depicts calibration curves for CD3 and CD45 quantification in dryblood spots (DBS);

FIG. 3 depicts TREC tested specimens and controls;

FIG. 4 depicts decoded TREC tested specimens;

FIGS. 5A and 5B depict an average calculated percent of original peptidesignal measured in Optical density by ELISA on Costar High bindingplates coated at 10 μg/ml and the sequences in the left hand column ofFIG. 5B correspond to SEQ ID NOS; 1 and 3-14 (from top to bottom) andthe sequences in the right hand column of FIG. 5B correspond to SEQ IDNOS; 2 and 15-24 (from top to bottom);

FIG. 6 depicts an IgG and IgY comparison where three plates with fourreplicates for each standard per plate were assayed in series, IgG isshown with open symbols and linear fit gave r2 values of 0.996 orgreater for all of the calibration curves;

FIG. 7 depicts calculated concentrations of T cells for the control andpositive specimens provided by The Danish National Newborn ScreeningBiobank; and

FIG. 8 depicts MFI and calculated concentrations of T cells for TheDanish National Newborn Screening Biobank specimens, positive for T celldeficiencies.

DETAILED DESCRIPTION

The present invention relates to novel peptides that would not producehigh affinity IgG antibody in mammalian hosts. In particular, theinvention relates to novel peptide sequences derived from chicken andhuman CD3 epsilon which would elicit not only an immune response butwould also allow for the development of high affinity IgY:

(SEQ ID NO: 1) 1) AKAKPVTRGAGA (12AA) (SEQ ID NO: 2) 2)LYSGLNQRRI (10AA)

The present invention also relates to novel peptide sequences thatcomprise, consist essentially of or consist of residues 8-12 of SEQ IDNO: 1 or residues 6-10 of SEQ ID NO: 2. The peptides of the presentinvention may also be useful for methods such as, but not limited to,ELISA, estimation of CD3+ cell counts, research, diagnosis,intracellular staining. The present invention relates to the productionof chicken antibodies, in particular, chicken antibodies derived fromSEQ ID NO: 1 and SEQ ID NO: 2 and/or peptides that comprise, consistessentially of or consist of residues 8-12 of SEQ ID NO: 1 or residues6-10 of SEQ ID NO: 2. Biologically, IgG antibodies have 3 bindingregions. Two regions are used to bind antigen (foreign debris or in thiscase CD3ε) and the third is used to bind different receptors for hostbiological functions. This can cause a problem with high background whenusing antibodies from other mammals to try and detect proteins fromhumans. This is not the case with chicken antibodies. Chicken antibodiesfunction the same as all the other IgG's, however, the third region onthe IgY is not active in humans, therefore background will be much lowercompared to antibodies produced in other animals.

Laying hens are highly cost-effective as producers of antibodiescompared with other mammals traditionally used for such production. Alsochicken antibodies have biochemical advantages over mammalian antibodiesdue to the phylogenetical differences between avian and mammalianspecies, resulting in increased sensitivity as well as decreasedbackground in immunological assays. In contrast to mammalian antibodies,chicken antibodies do not activate the human complement system nor willthey react with rheumatoid factors, human anti-mouse IgG antibodies, orbacterial and human Fc receptors. Thus chicken antibodies offer manyadvantages over mammalian antibodies and are, even now, beginning toreplace conventional sources of custom produced antibodies (see, e.g.,http://www.oramune.com/custom.aspx?id=7).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, and immunology, which are within the skillof the art. Such techniques are explained fully in the literature. See,e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Press; DNA Cloning, Vols. I and II (D. N. Glovered. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal CellCulture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRLpress, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984);the series, Methods In Enzymology (S. Colowick and N. Kaplan eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell ScientificPublications).

As used herein, the term “antigen” or “immunogen” means a substance thatinduces a specific immune response in a host animal. The antigen maycomprise a whole organism, killed, attenuated or live; a subunit orportion of an organism; a recombinant vector containing an insertexpressing an epitope, polypeptide, peptide, protein, or fragmentthereof with immunogenic properties; a piece or fragment of nucleic acidcapable of inducing an immune response upon presentation to a hostanimal; a protein, a polypeptide, a peptide, an epitope, a hapten, orany combination thereof. Alternately, the immunogen or antigen maycomprise a toxin or antitoxin.

The term “immunogenic protein or peptide” as used herein also includespeptides and polypeptides that are immunologically active in the sensethat once administered to the host, it is able to evoke an immuneresponse of the humoral and/or cellular type directed against theprotein. Preferably the protein fragment is such that it hassubstantially the same immunological activity as the total protein.Thus, a protein fragment according to the invention comprises orconsists essentially of or consists of at least one epitope or antigenicdeterminant. The term epitope, also known as antigenic determinant, isthe part of a macromolecule recognized by the immune system and able toinduce an immune reaction of the humoral type (B cells) and/or cellulartype (T cells).

The term “immunogenic protein or peptide” further contemplatesdeletions, additions and substitutions to the sequence, so long as thepolypeptide functions to produce an immunological response as definedherein. In this regard, particularly preferred substitutions willgenerally be conservative in nature, i.e., those substitutions that takeplace within a family of amino acids. For example, amino acids aregenerally divided into four families: (1) acidic—aspartate andglutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine,cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified as aromatic amino acids. It isreasonably predictable that an isolated replacement of leucine withisoleucine or valine, or vice versa; an aspartate with a glutamate orvice versa; a threonine with a serine or vice versa; or a similarconservative replacement of an amino acid with a structurally relatedamino acid, will not have a major effect on the biological activity.Proteins having substantially the same amino acid sequence as thereference molecule but possessing minor amino acid substitutions that donot substantially affect the immunogenicity of the protein are,therefore, within the definition of the reference polypeptide.

The term epitope is the part of a macromolecule recognized by the immunesystem and able to induce an immune reaction of the humoral type (Bcells) and/or cellular type (T cells) The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite”. Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to a composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, and/or cytotoxic T cells, directed specifically to an antigen orantigens included in the composition or vaccine of interest.

Preferably, the host will display either a therapeutic or protectiveimmunological response such that resistance to new infection will beenhanced and/or the clinical severity of the disease reduced. Suchprotection will be demonstrated by either a reduction or lack ofsymptoms normally displayed by an infected host, a quicker recovery timeand/or a lowered viral titer in the infected host.

The term“immunogenic” protein or polypeptide as used herein also refersto an amino acid sequence which elicits an immunological response asdescribed above. An “immunogenic” protein or polypeptide, as usedherein, includes the full-length sequence of the protein, analogsthereof, or immunogenic fragments thereof. By “immunogenic fragment” ismeant a fragment of a protein which includes one or more epitopes andthus elicits the immunological response described above. Such fragmentscan be identified using any number of epitope mapping techniques, wellknown in the art. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996). For example,linear epitopes may be determined by e.g., concurrently synthesizinglarge numbers of peptides on solid supports, the peptides correspondingto portions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al., 1984; Geysen et al., 1986, all incorporatedherein by reference in their entireties. Similarly, conformationalepitopes are readily identified by determining spatial conformation ofamino acids such as by, e.g., x-ray crystallography and 2-dimensionalnuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.Methods especially applicable to the proteins of T. parva are fullydescribed in the PCT Application Serial No. PCT/US2004/022605incorporated herein by reference in its entirety.

Synthetic antigens are also included within the definition, for example,polyepitopes, flanking epitopes, and other recombinant or syntheticallyderived antigens. See, e.g., Bergmann et al., 1993; Bergmann et al.,1996; Suhrbier, 1997; Gardner et al., 1998. Immunogenic fragments, forpurposes of the present invention, will usually include at least about 3amino acids, preferably at least about 5 amino acids, more preferably atleast about 10-15 amino acids, and most preferably about 15-25 aminoacids or more amino acids, of the molecule. There is no critical upperlimit to the length of the fragment, which could comprise nearly thefull-length of the protein sequence, or even a fusion protein comprisingat least one epitope of the protein.

Accordingly, a minimum structure of a polynucleotide expressing anepitope is that it comprises or consists essentially of or consists ofnucleotides to encode an epitope or antigenic determinant of a CD3epsilon protein or polyprotein. A polynucleotide encoding a fragment ofthe total protein or polyprotein, more advantageously, comprises orconsists essentially of or consists of a minimum of 15 nucleotides,advantageously about 30-45 nucleotides, and preferably about 45-75, atleast 57, 87 or 150 consecutive or contiguous nucleotides of thesequence encoding the total protein or polyprotein. Epitopedetermination procedures, such as, generating overlapping peptidelibraries (Hemmer et al., 1998), Pepscan (Geysen et al., 1984; Geysen etal., 1985; Van der Zee R. et al., 1989; Geysen, 1990; Multipin®. PeptideSynthesis Kits de Chiron) and algorithms (De Groot et al., 1999), and inPCT Application Serial No. PCT/US2004/022605 all of which areincorporated herein by reference in their entireties can be used in thepractice of the invention, without undue experimentation. Otherdocuments cited and incorporated herein may also be consulted formethods for determining epitopes of an immunogen or antigen and thusnucleic acid molecules that encode such epitopes.

A “polynucleotide” is a polymeric form of nucleotides of any length,that contains deoxyribonucleotides, ribonucleotides, and analogs in anycombination. Polynucleotides may have three-dimensional structure, andmay perform any function, known or unknown. The term “polynucleotide”includes double-, single-stranded, and triple-stranded helicalmolecules. Unless otherwise specified or required, any embodiment of theinvention described herein that is a polynucleotide encompasses both thedouble stranded form and each of two complementary forms known orpredicted to make up the double stranded form of either the DNA, RNA orhybrid molecule.

The term “codon optimization” refers to the process of optimallyconfiguring the nucleic acid sequence encoding a protein, polypeptide,antigen, epitope, domain or fragment for expression/translation inselected host. In general, gene expression levels depend on manyfactors, such as promoter sequences and regulatory elements. One of themost important factors is the adaptation of the codon usage of thetranscript gene to the typical codon usage of the host (Lithwich, G. andMargalit, H., Genome Res. 13, 2665-2673, 2003). Therefore, highlyexpressed genes in prokaryotic genomes under translational selectionhave a pronounced codon usage bias. This is because they use a smallsubset of codons that are recognized by the most abundant tRNA species(Ikemura, T., J. Mol. Biol. 151, 389-409, 1981). The force thatmodulates this codon adaptation is called translational selection andits strength is important in fast-growing bacteria (Rocha, E. P., GenomeRes. 14, 2279-2286, 2004; Sharp, P. M. et al., Nucleic Acids Res. 33,1141-1153)). If a gene contains codons that are rarely used by the host,its expression level will not be maximal. This may be one of thelimitations of heterologous protein expression (Gustafsson, C. et al.,Trends Biotechnol. 22, 346-353, 2004) and the development of DNAvaccines (Ivory, C. and Chadee, K., Genet. Vaccines Ther. 2, 17, 2004).A high number of synthetic genes have been re-designed to increase theirexpression level. The Synthetic Gene Database (SGDB) (Wu, G. et al.,Nucleic Acids Res. 35, D76-D79, 2007) contains information from morethan 200 published experiments on synthetic genes. In the design processof a nucleic acid sequence that will be inserted into a new host toexpress a certain protein in optimal amounts, codon usage optimizationis usually one of the first steps (Gustafsson, C., Trends Biotechnol.22, 346-353, 2004). Codon usage optimization basically involves alteringthe rare codons in the target gene so that they more closely reflect thecodon usage of the host without modifying the amino acid sequence of theencoded protein (Gustafsson, C., Trends Biotechnol. 22, 346-353, 2004).The information usually used for the optimization process is thereforethe DNA or protein sequence to be optimized and a codon usage table(reference set) of the host.

There are several public web servers and stand-alone applications thatallow some kind of codon optimization by anyone skilled in the art.‘GeneDesign’ (Richardson, S. M. et al., Genome Res. 16, 550-556, 2006),‘Synthetic Gene Designer’ (Wu, G. et al., Protein Expr. Purif. 47,441-445, 2006) and ‘Gene Designer’ (Villalobos, A. et al., BMCBioinformatics 7, 285, 2006) are packages that provide a platform forsynthetic gene design, including a codon optimization step. With regardto the methods for codon usage optimization available in each server orprogram, the first programs developed used only the ‘one amino acid—onecodon’ approach. More recent programs and servers now include furthermethods to create some codon usage variability. This variabilityreflects the codon usage variability of natural highly expressed genesand enables additional criteria to be introduced (such as the avoidanceof restriction sites) in the optimization process. Most applications andweb servers described herein provide three methods of codonoptimization: a complete optimization of all codons, an optimizationbased on the relative codon usage frequencies of the reference set thatuses a Monte Carlo approach and a novel approach designed to maximizethe optimization with the minimum changes between the query andoptimized sequences.

The following are non-limiting examples of polynucleotides: a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, siRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthiolate, and nucleotide branches. The sequence of nucleotides may befurther modified after polymerization, such as by conjugation, with alabeling component. Other types of modifications included in thisdefinition are caps, substitution of one or more of the naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides or solid support. The polynucleotidescan be obtained by chemical synthesis or derived from a microorganism.

The invention further comprises a complementary strand to apolynucleotide encoding a CD3 epsilon protein, antigen, epitope orimmunogen. The complementary strand can be polymeric and of any length,and can contain deoxyribonucleotides, ribonucleotides, and analogs inany combination thereof.

The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer can be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

An “isolated” polynucleotide or polypeptide is one that is substantiallyfree of the materials with which it is associated in its nativeenvironment. By substantially free, is meant at least 50%,advantageously at least 70%, more advantageously at least 80%, and evenmore advantageously at least 90% or at least 95% free of thesematerials.

Hybridization reactions can be performed under conditions of different“stringency.” Conditions that increase stringency of a hybridizationreaction are well known. See for example, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989). Examples ofrelevant conditions include (in order of increasing stringency):incubation temperatures of 25° C., 37° C., 50° C., and 68° C.; bufferconcentrations of 10×SSC, 6×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 MNaCl and 15 mM citrate buffer) and their equivalent using other buffersystems; formamide concentrations of 0%, 25%, 50%, and 75%; incubationtimes from 5 minutes to 24 hours; 1, 2 or more washing steps; washincubation times of 1, 2, or 15 minutes; and wash solutions of 6×SSC,1×SSC, 0.1×SSC, or deionized water.

The invention further encompasses polynucleotides encoding functionallyequivalent variants and derivatives of the CD3 epsilon polypeptides andfunctionally equivalent fragments thereof that may enhance, decrease ornot significantly affect inherent properties of the polypeptides encodedthereby. These functionally equivalent variants, derivatives, andfragments display the ability to retain CD3 epsilon activity. Forinstance, changes in a DNA sequence that do not change the encoded aminoacid sequence, as well as those that result in conservativesubstitutions of amino acid residues, one or a few amino acid deletionsor additions, and substitution of amino acid residues by amino acidanalogs are those which will not significantly affect properties of theencoded polypeptide. Conservative amino acid substitutions areglycine/alanine; valine/isoleucine/leucine; asparagine/glutamine;aspartic acid/glutamic acid; serine/threonine/methionine;lysine/arginine; and phenylalanine/tyrosine/tryptophan. In oneembodiment, the variants have at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% homology oridentity to the CD3 epsilon polynucleotide or polypeptide of interest.

For the purposes of the present invention, sequence identity or homologyis determined by comparing the sequences when aligned so as to maximizeoverlap and identity while minimizing sequence gaps. In particular,sequence identity may be determined using any of a number ofmathematical algorithms. A non-limiting example of a mathematicalalgorithm used for comparison of two sequences is the algorithm ofKarlin et al., 1990 modified as in Karlin et al., 1993.

Another example of a mathematical algorithm used for comparison ofsequences is the algorithm of Myers et al., 1988. Such an algorithm isincorporated into the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Yet another useful algorithm for identifying regions of local sequencesimilarity and alignment is the FASTA algorithm as described in Pearsonet al., 1988.

Advantageous for use according to the present invention is the WU-BLAST(Washington University BLAST) version 2.0 software. WU-BLAST version 2.0executable programs for several UNIX platforms can be downloaded fromftp://blast.wustl.edu/blast/executables. This program is based onWU-BLAST version 1.4, which in turn is based on the public domainNCBI-BLAST version 1.4 (Altschul et al., 1996; Altschul et al., 1990;Gish et al., 1993; Karlin et al., 1993; all of which are incorporated byreference herein).

In general, comparison of amino acid sequences is accomplished byaligning an amino acid sequence of a polypeptide of a known structurewith the amino acid sequence of a polypeptide of unknown structure.Amino acids in the sequences are then compared and groups of amino acidsthat are homologous are grouped together. This method detects conservedregions of the polypeptides and accounts for amino acid insertions anddeletions. Homology between amino acid sequences can be determined byusing commercially available algorithms (see also the description ofhomology above). In addition to those otherwise mentioned herein,mention is made too of the programs BLAST, gapped BLAST, BLASTN, BLASTP,and PSI-BLAST, provided by the National Center for BiotechnologyInformation. These programs are widely used in the art for this purposeand can align homologous regions of two amino acid sequences.

In all search programs in the suite the gapped alignment routines areintegral to the database search itself. Gapping can be turned off ifdesired. The default penalty (Q) for a gap of length one is Q=9 forproteins and BLASTP, and Q=10 for BLASTN, but may be changed to anyinteger. The default per-residue penalty for extending a gap (R) is R=2for proteins and BLASTP, and R=10 for BLASTN, but may be changed to anyinteger. Any combination of values for Q and R can be used in order toalign sequences so as to maximize overlap and identity while minimizingsequence gaps. The default amino acid comparison matrix is BLOSUM62, butother amino acid comparison matrices such as PAM can be utilized.

Alternatively or additionally, the term “homology” or “identity”, forinstance, with respect to a nucleotide or amino acid sequence, canindicate a quantitative measure of homology between two sequences. Thepercent sequence homology can be calculated as(N_(ref)−N_(dif))*100/N_(ref), wherein N_(dif) is the total number ofnon-identical residues in the two sequences when aligned and whereinN_(ref) is the number of residues in one of the sequences. Hence, theDNA sequence AGTCAGTC will have a sequence identity of 75% with thesequence AATCAATC (N_(ref)=8; N_(dif)=2).

Alternatively or additionally, “homology” or “identity” with respect tosequences can refer to the number of positions with identicalnucleotides or amino acids divided by the number of nucleotides or aminoacids in the shorter of the two sequences wherein alignment of the twosequences can be determined in accordance with the Wilbur and Lipmanalgorithm (Wilbur & Lipman, Proc Natl Acad Sci USA. 1983 February;80(3):726-30, incorporated herein by reference), for instance, using awindow size of 20 nucleotides, a word length of 4 nucleotides, and a gappenalty of 4, and computer-assisted analysis and interpretation of thesequence data including alignment can be conveniently performed usingcommercially available programs (e.g., Vector NTI Software™, InvitrogenInc. CA). When RNA sequences are said to be similar, or have a degree ofsequence identity or homology with DNA sequences, thymidine (T) in theDNA sequence is considered equal to uracil (U) in the RNA sequence.Thus, RNA sequences are within the scope of the invention and can bederived from DNA sequences, by thymidine (T) in the DNA sequence beingconsidered equal to uracil (U) in RNA sequences.

And, without undue experimentation, the skilled artisan can consult withmany other programs or references for determining percent homology.

The invention further encompasses the CD3 epsilon polynucleotidescontained in a vector molecule or an expression vector and operablylinked to a promoter element and optionally to an enhancer.

A “vector” refers to a recombinant DNA or RNA plasmid, bacteriophage, orvirus that comprises a heterologous polynucleotide to be delivered to atarget cell, either in vitro or in vivo. The heterologous polynucleotidemay comprise a sequence of interest for purposes of prevention ortherapy, and may optionally be in the form of an expression cassette. Asused herein, a vector needs not be capable of replication in theultimate target cell or subject. The term includes vectors for cloningas well as viral vectors.

The term “recombinant” means a polynucleotide of semisynthetic, orsynthetic origin that either does not occur in nature or is linked toanother polynucleotide in an arrangement not found in nature.

“Heterologous” means derived from a genetically distinct entity from therest of the entity to which it is being compared. For example, apolynucleotide, may be incorporated by genetic engineering techniquesinto a plasmid or vector derived from a different source, and is thus aheterologous polynucleotide. A promoter removed from its native codingsequence and operatively linked to a coding sequence other than thenative sequence is a heterologous promoter.

The polynucleotides of the invention may comprise additional sequences,such as additional encoding sequences within the same transcriptionunit, controlling elements such as promoters, ribosome binding sites,5′UTR, 3′UTR, transcription terminators, polyadenylation sites,additional transcription units under control of the same or a differentpromoter, sequences that permit cloning, expression, homologousrecombination, and transformation of a host cell, and any such constructas may be desirable to provide embodiments of this invention.

Elements for the expression of a CD3 epsilon polypeptide, antigen,epitope or immunogen are advantageously present in an inventive vector.In minimum manner, this comprises, consists essentially of, or consistsof an initiation codon (ATG), a stop codon and a promoter, andoptionally also a polyadenylation sequence for certain vectors such asplasmid and certain viral vectors, e.g., viral vectors other thanpoxviruses. When the polynucleotide encodes a polyprotein fragment, e.g.a CD3 epsilon peptide, advantageously, in the vector, an ATG is placedat 5′ of the reading frame and a stop codon is placed at 3′. Otherelements for controlling expression may be present, such as enhancersequences, stabilizing sequences, such as intron and signal sequencespermitting the secretion of the protein.

Methods for making and/or administering a vector or recombinants orplasmid for expression of gene products of genes of the invention eitherin vivo or in vitro can be any desired method, e.g., a method which isby or analogous to the methods disclosed in, or disclosed in documentscited in: U.S. Pat. Nos. 4,603,112; 4,769,330; 4,394,448; 4,722,848;4,745,051; 4,769,331; 4,945,050; 5,494,807; 5,514,375; 5,744,140;5,744,141; 5,756,103; 5,762,938; 5,766,599; 5,990,091; 5,174,993;5,505,941; 5,338,683; 5,494,807; 5,591,639; 5,589,466; 5,677,178;5,591,439; 5,552,143; 5,580,859; 6,130,066; 6,004,777; 6,130,066;6,497,883; 6,464,984; 6,451,770; 6,391,314; 6,387,376; 6,376,473;6,368,603; 6,348,196; 6,306,400; 6,228,846; 6,221,362; 6,217,883;6,207,166; 6,207,165; 6,159,477; 6,153,199; 6,090,393; 6,074,649;6,045,803; 6,033,670; 6,485,729; 6,103,526; 6,224,882; 6,312,682;6,348,450; 6,312,683, and 6,596,279; U.S. patent application Serial No.920,197, filed Oct. 16, 1986; WO 90/01543; WO91/11525; WO 94/16716; WO96/39491; WO 98/33510; EP 265785; EP 0 370 573; Andreansky et al., 1996;Ballay et al., 1993; Feigner et al., 1994; Frolov et al., 1996; Graham,1990; Grunhaus et al., 1992; Ju et al., 1998; Kitson et al., 1991;McClements et al., 1996; Moss, 1996; Paoletti, 1996; Pennock et al.,1984; Richardson (Ed), 1995; Smith et al., 1983; Robertson et al., 1996;Robinson et al., 1997; and Roizman, 1996. Thus, the vector in theinvention can be any suitable recombinant virus or virus vector, such asa poxvirus (e.g., vaccinia virus, avipox virus, canarypox virus, fowlpoxvirus, raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., humanadenovirus, canine adenovirus), herpesvirus (e.g. canine herpesvirus),baculovirus, retrovirus, etc. (as in documents incorporated herein byreference); or the vector can be a plasmid. The herein cited andincorporated herein by reference documents, in addition to providingexamples of vectors useful in the practice of the invention, can alsoprovide sources for non-CD3 epsilon peptides or fragments thereof to beexpressed by vector or vectors in, or included in, the compositions ofthe invention.

The present invention also relates to preparations comprising vectors,such as expression vectors, e.g., therapeutic compositions. Thepreparations can comprise, consist essentially of, or consist of one ormore vectors, e.g., expression vectors, such as in vivo expressionvectors, comprising, consisting essentially or consisting of (andadvantageously expressing) one or more of CD3 epsilon polypeptides,antigens, epitopes or immunogens. Advantageously, the vector containsand expresses a polynucleotide that includes, consists essentially of,or consists of a polynucleotide coding for (and advantageouslyexpressing) a CD3 epsilon antigen, epitope or immunogen, in apharmaceutically acceptable carrier, excipient or vehicle. Thus,according to an embodiment of the invention, the other vector or vectorsin the preparation comprises, consists essentially of or consists of apolynucleotide that encodes, and under appropriate circumstances thevector expresses one or more other proteins of a CD3 epsilonpolypeptide, antigen, epitope or immunogen (e.g., hemagglutinin,neuraminidase, nucleoprotein) or a fragment thereof.

According to another embodiment, the vector or vectors in thepreparation comprise, or consist essentially of, or consist ofpolynucleotide(s) encoding one or more proteins or fragment(s) thereofof a CD3 epsilon polypeptide, antigen, epitope or immunogen, the vectoror vectors expressing the polynucleotide(s). The inventive preparationadvantageously comprises, consists essentially of, or consists of, atleast two vectors comprising, consisting essentially of, or consistingof, and advantageously also expressing, advantageously in vivo underappropriate conditions or suitable conditions or in a suitable hostcell, polynucleotides from different CD3 epsilon isolates encoding thesame proteins and/or for different proteins, but advantageously the sameproteins. Preparations containing one or more vectors containing,consisting essentially of or consisting of polynucleotides encoding, andadvantageously expressing, advantageously in vivo, a CD3 epsilonpolypeptide, antigen, fusion protein or an epitope thereof. Theinvention is also directed at mixtures of vectors that contain, consistessentially of, or consist of coding for, and express, different CD3epsilon proteins, polypeptides, antigens, epitopes or immunogens, e.g.,a D3 epsilon polypeptide, antigen, epitope or immunogen from differentspecies such as, but not limited to, humans, horses, pigs, seals,whales, in addition to avian species including chicken, turkeys, ducksand geese.

The term plasmid covers any DNA transcription unit comprising apolynucleotide according to the invention and the elements necessary forits in vivo expression in a cell or cells of the desired host or target;and, in this regard, it is noted that a supercoiled plasmid and all ofits topoisomers, open-circular plasmid, as well as linear forms of theplasmid, are intended to be within the scope of the invention.

Each plasmid comprises or contains or consists essentially of, inaddition to the heterologous polynucleotide encoding a recombinantprotein, antigen, epitope or immunogen, optionally fused with apolynucleotide encoding an heterologous peptide sequence, variant,analog or fragment, operably linked to a promoter or under the controlof a promoter or dependent upon a promoter. In general, it isadvantageous to employ a strong promoter that is functional ineukaryotic cells. The preferred strong promoter is the immediate earlycytomegalovirus promoter (CMV-IE) of human or murine origin, oroptionally having another origin such as the rat or guinea pig. TheCMV-IE promoter can comprise the actual promoter segment, which may ormay not be associated with the enhancer segment. Reference can be madeto EP-A-260 148, EP-A-323 597, U.S. Pat. Nos. 5,168,062, 5,385,839, and4,968,615, as well as to PCT Application No WO87/03905. The CMV-IEpromoter is advantageously a human CMV-IE (Boshart et al., 1985) ormurine CMV-IE.

In more general terms, the promoter is either of a viral or a cellularorigin. A strong viral promoter other than CMV-IE that may be usefullyemployed in the practice of the invention is the early/late promoter ofthe SV40 virus or the LTR promoter of the Rous sarcoma virus. A strongcellular promoter that may be usefully employed in the practice of theinvention is the promoter of a gene of the cytoskeleton, such as e.g.the desmin promoter (Kwissa et al., 2000), or the actin promoter(Miyazaki et al., 1989).

Functional sub fragments of these promoters, i.e., portions of thesepromoters that maintain an adequate promoting activity, are includedwithin the present invention, e.g. truncated CMV-IE promoters accordingto PCT Application No. WO98/00166 or U.S. Pat. No. 6,156,567 can be usedin the practice of the invention. A promoter in the practice of theinvention consequently includes derivatives and sub fragments of afull-length promoter that maintain an adequate promoting activity andhence function as a promoter, preferably promoting activitysubstantially similar to that of the actual or full-length promoter fromwhich the derivative or sub fragment is derived, e.g., akin to theactivity of the truncated CMV-IE promoters of U.S. Pat. No. 6,156,567 tothe activity of full-length CMV-IE promoters. Thus, a CMV-IE promoter inthe practice of the invention can comprise or consist essentially of orconsist of the promoter portion of the full-length promoter and/or theenhancer portion of the full-length promoter, as well as derivatives andsub fragments.

Preferably, the plasmids comprise or consist essentially of otherexpression control elements. It is particularly advantageous toincorporate stabilizing sequence(s), e.g., intron sequence(s),preferably the first intron of the hCMV-IE (PCT Application No.WO89/01036), the intron II of the rabbit β13-globin gene (van Ooyen etal., 1979).

As to the polyadenylation signal (polyA) for the plasmids and viralvectors other than poxviruses, use can more be made of the poly(A)signal of the bovine growth hormone (bGH) gene (see U.S. Pat. No.5,122,458), or the poly(A) signal of the rabbit β-globin gene or thepoly(A) signal of the SV40 virus.

According to another embodiment of the invention, the expression vectorsare expression vectors used for the in vitro expression of proteins inan appropriate cell system. The expressed proteins can be harvested inor from the culture supernatant after, or not after secretion (if thereis no secretion a cell lysis typically occurs or is performed),optionally concentrated by concentration methods such as ultrafiltrationand/or purified by purification means, such as affinity, ion exchange orgel filtration-type chromatography methods.

A “host cell” denotes a prokaryotic or eukaryotic cell that has beengenetically altered, or is capable of being genetically altered byadministration of an exogenous polynucleotide, such as a recombinantplasmid or vector. When referring to genetically altered cells, the termrefers both to the originally altered cell and to the progeny thereof.Advantageous host cells include, but are not limited to, baby hamsterkidney (BHK) cells, colon carcinoma (Caco-2) cells, COST cells, MCF-7cells, MCF-10A cells, Madin-Darby canine kidney (MDCK) lines, mink lung(Mv1Lu) cells, MRC-5 cells, U937 cells and VERO cells. Polynucleotidescomprising a desired sequence can be inserted into a suitable cloning orexpression vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification. Polynucleotidescan be introduced into host cells by any means known in the art. Thevectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingdirect uptake, endocytosis, transfection, f-mating, electroporation,transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is infectious,for instance, a retroviral vector). The choice of introducing vectors orpolynucleotides will often depend on features of the host cell.

In an advantageous embodiment, the invention provides for theadministration of a therapeutically effective amount of a formulationfor the delivery and expression of a protein, antigen, epitope orimmunogen in a target cell. Determination of the therapeuticallyeffective amount is routine experimentation for one of ordinary skill inthe art. In one embodiment, the formulation comprises an expressionvector comprising a polynucleotide that expresses a CD3 epsilon antigen,epitope or immunogen and a pharmaceutically acceptable carrier, vehicleor excipient. In an advantageous embodiment, the pharmaceuticallyacceptable carrier, vehicle or excipient facilitates transfection and/orimproves preservation of the vector or protein.

The pharmaceutically acceptable carriers or vehicles or excipients arewell known to one skilled in the art. For example, a pharmaceuticallyacceptable carrier or vehicle or excipient can be sterile water, a 0.9%NaCl (e.g., saline) solution or a phosphate buffer. Otherpharmaceutically acceptable carriers or vehicles or excipients that canbe used for methods of this invention include, but are not limited to,poly-(L-glutamate) or polyvinylpyrrolidone. The pharmaceuticallyacceptable carriers or vehicles or excipients may be any compound orcombination of compounds facilitating the administration of the vector(or protein expressed from an inventive vector in vitro);advantageously, the carrier, vehicle or excipient may facilitatetransfection and/or improve preservation of the vector (or protein).Doses and dose volumes are herein discussed in the general descriptionand can also be determined by the skilled artisan from this disclosureread in conjunction with the knowledge in the art, without any undueexperimentation.

The present invention also encompasses methods for making antibodies. Inan advantageous embodiment, the antibodies are chicken antibodies. In aparticularly advantageous embodiment, the antibodies are isolated fromthe yolk of eggs laid by immunized hens. A laying hen producesapproximately five to six eggs per week with a yolk volume ofapproximately 15 ml per egg, the antibody concentration of which iscomparable to that of serum. Therefore, in one week a hen produces eggantibodies equivalent to 75 to 90 ml of serum or 150 to 180 ml of wholeblood. This could be compared to an immunized rabbit, which yieldsapproximately 20 ml whole blood per week. Only large mammals such ascows or horses can produce more antibodies than a laying hen. The bloodcollection procedure is time consuming and stressful for the animal.Furthermore, the cost of feeding and handling is considerably lower fora hen than for a rabbit. Crude egg yolk may be used as an antibodysource, but the lipids in the yolk may interfere with the antibodyactivity. Therefore, avian antibodies may be purified from the yolkprior to use.

To stimulate the immune response of experimental animals, the desiredantigen is applied in combination with various adjuvant compounds(Hilgers et al. 1998, Vet. Immunol. Immunopathol. 66 159-171). Usingthese compounds, antigen is appropriately presented, e.g. in the form ofan emulsion, to the organism and the immune system non-specificallystimulated to produce antibodies by additional components (e.g.adamantyldipeptide, inactivated microorganisms or their parts). Oftested adjuvant preparations the best results were obtained withemulsions of antigens in mineral oil mixed with complete Freund'sadjuvant for the first injection and with incomplete Freund's forboosters. Similar immune responses were found when only solutions ofantigens in PBS were used for injections (Svendsen-Bollen et al. 1996,J. Immunol. Methods 191 113-120, Schwarzkopf & Thiele 1996, ALTEX 13(Suppl. 96), 22-25). Just recently, application of lipid nanoparticlescausing only minor tissue irritation at the injection sites, appears tobe a promising alternative to complete Freund's adjuvant (Olbrich et al.2002, ATLA 30 443-458).

In respect to antibody titers it is hard to judge which organism, therabbit or the chicken, is superior for antibody production. Even whenthe experimental conditions are kept to be the same for both animalspecies the titers depend on the antigen immunogenicity for the animalused. For example, the chicken is able to produce antibodies against oneserotype of rotavirus with a neutralization titer more than 4 timeshigher than that derived from rabbit blood. On the other hand, foranother serotype chicken antibodies show lower activity than rabbitantisera (Hatta et al. 1993, Biosci. Biotech. Biochem. 57 450-454).Thanks to the evolutionary distance between birds and mammals, thechicken is superior for the production of antibodies against conservedmammalian antigens, which are hardly immunogenic for experimentalmammals. Chicken IgY is usually produced against a greater number ofantigenic epitopes on a mammalian antigen thus giving an amplifiedsignal and greater test sensitivity. Another advantage lies in thepossibility of developing high titer chicken antibodies even though lowdoses of mammalian antigen (0.001-0.01 mg/dose) are applied (Gassmann etal. 1990, FASEB J. 4 2528-2532, Larsson et al. 1998, Food Agric.Immunol. 10 29-36, Knecht et al. 1996, Eur. J. Biochem. 236 609-613).

So far, the major limitation preventing a wide application of IgYs liesmost probably in their purification from egg yolks. It is true that aprocedure as simple as the preparation of antisera from mammalian bloodis not available for chicken antibodies. IgY comprises about 5% of eggyolk proteins dispersed in yolk lipid emulsion together withlipoproteins and glycoproteins (Juneja & Kim, 1997, Hen Eggs: TheirBasic and Applied Science pp. 57-72 eds T Yamamoto, L R Juneja, H Hattaand M Kim (USA: CRC Press)). There are plenty of different proceduresdeveloped for IgY purification (Hodek et al. 1998, CZ Patent 281298,Stalberg et al. 2001, De Meulenaer & Huyghebaert 2001). The first stepof these procedures (after yolk separation) is always based on removalof the lipid fraction by its extraction into organic solvent,precipitation using freezing or precipitation agents or hydrophobicchromatography. Recently, the use of an aqueous two-phase system withphosphate and Triton X-100 separation of lipids and watersolubleproteins (IgY fraction) has been introduced (Stalberg et al. 2001, Ups.J. Med. Sci. 106 99-110). The resulting water-soluble protein fractionis usually separated by fraction precipitation or chromatography onion-exchange, thiophilic or size-exclusion columns (Polson et al. 1980,Immunol. Commun. 9 495-514, Bade & Stegemann 1984, J. Immunol. Methods72 421-426, Hassl & Aspöck 1988, J. Immunol. Methods 110 225-228, Hattaet al. 1990, Agric. Biol. Chem. 54 2531-2535, Akita & Nakai 1993, J.Immunol. Methods 160 207-214, Schwarzkopf & Thiele 1996, ALTEX 13(Suppl. 96), 22-25, Cook et al. 2001, J. Biosci. Bioeng. 91 305-310).

Interestingly, chicken antibodies were efficiently captured from crudesamples on an affinity column with immobilized synthetic ligand forimmunoglobulins. Using this technique in a single purification step thepurity of IgY higher than 90% was obtained (Verdoliva et al. 2000, J.Chromatogr. 749 233-242). The majority of protocols, however, apply 2-3purification steps to obtain a final preparation of a high purity (98%),yielding 70-100 mg IgY per yolk. To prepare monospecific antibodies, anaffinity chromatography technique on immobilized antigen is usuallyexploited. Specifically bound IgY is eluted by strong acidic or basicbuffers (Ntakarutimana et al. 1992, J. Immunol. Methods 153 133-140,Kuronen et al. 1997, Eur. J. Clin. Chem. Clin. Biochem. 35 435-440, Tiniet al. 2002, Comp. Biochem. Physiol. A Mol. Integr. Physiol. 31569-574). Purified IgYs show high stability when they are stored at 4°C. They have retained their activity for more than 10 years (Larsson etal. 1999, Food Agric. Immunol. 11 43-49).

Chloroform/PEG extraction may be performed on yolks for IgYpurification, after which the IgY may be further manipulated to enrichfor the Peptide specific IgY.

The present invention also encompasses methods for diagnostic andgenetic analysis of SCID markers, such as CD3 and CD45. In anadvantageous embodiment, the analysis may comprise the steps ofconstructing an appropriately labeled beadset, exposing the headset to aclinical sample, and analyzing the combined sample/beadset by flowcytometry is disclosed. Flow cytometric measurements are used toclassify, in real-time, beads within an exposed beadset and textualexplanations, based on the accumulated data obtained during real-timeanalysis, are generated for the user.

One well known prior art technique used in assay procedures for which amultiplexed assay capability would be particularly advantageous is flowcytometry. Flow cytometry is an optical technique that analyzesparticular particles in a fluid mixture based on the particles′ opticalcharacteristics using an instrument known as a flow cytometer.Background information on flow cytometry may be found in Shapiro,“Practical Flow Cytometry,” Third Ed. (Alan R. Liss, Inc. 1995); andMelamed et al., “Flow Cytometry and Sorting,” Second Ed. (Wiley-Liss1990), which are incorporated herein by reference. Flow cytometershydrodynamically focus a fluid suspension of particles into a thinstream so that the particles flow down the stream in substantiallysingle file and pass through an examination zone. A focused light beam,such as a laser beam illuminates the particles as they flow through theexamination zone. Optical detectors within the flow cytometer measurecertain characteristics of the light as it interacts with the particles.Commonly used flow cytometers such as the Becton-DickinsonImmunocytometry Systems “FACSCAN” (San Jose, Calif.) can measure forwardlight scatter (generally correlated with the refractive index and sizeof the particle being illuminated), side light scatter (generallycorrelated with the particle's size), and particle fluorescence at oneor more wavelengths. (Fluorescence is typically imparted byincorporating, or attaching a fluorochrome within the particle.) Flowcytometers and various techniques for their use are described,generally, in “Practical Flow Cytometry” by Howard M. Shapiro (Alan R.Liss, Inc., 1985) and “Flow Cytometry and Sorting, Second Edition”edited by Melamed et al. (Wiley-Liss, 1990).

An important feature of the flow cytometric technology and techniquesdescribed here is the fabrication and use of particles (e.g.,microspheres or beads that make up a headset). It is through the use ofappropriately labeled homogeneous bead subsets, combined to produce apooled beadset, that the instant multiplexed assay method is practiced.Beads suitable for use as a starting material in accordance with theinvention are generally known in the art and may be obtained frommanufacturers such as Luminex, Spherotech and Molecular Probes. Once ahomogeneous subset of beads is obtained, the beads are labeled with anappropriate reactant such as a biomolecule (such as an antibody), DNAsequence, and/or other reactant. Known methods to incorporate suchlabels include polymerization, dissolving, and attachment.

Development of a multiplexed assay for use in accordance with theinvention can be divided into three phases: (1) preprocessing, (2)real-time analysis, and (3) interpretation. During the preprocessingphase, baseline data is collected independently, via flow cytometrictechniques, for each of an assay's bead subsets. Baseline data is usedto generate a set of functions that can classify any individual bead asbelonging to one of the assay's subsets or to a rejection class. Duringthe analysis phase, flow cytometric measurements are used to classify,in real-time, each bead within an exposed beadset according to theaforementioned functions. Additionally, measurements relating to eachsubset's analyte are accumulated. During the interpretation phase theassay's real-time numerical results are associated with textualexplanations and these textual explanations are displayed to a user. Theinventive method allows the detection of a plurality of analytessimultaneously during a single flow cytometric processing step. Benefitsof the inventive multiplex assay method include increased speed andreduced cost to analyze a clinical sample.

In particular, the assays developed by Luminex are particularlyadvantageous for the present invention, e.g., the disclosures of U.S.Pat. Nos. 7,645,868; 7,608,398; 7,551,763; 7,523,637; 7,505,131;7,465,540; 7,455,980; 7,445,844; 7,385,053; 7,362,432; 7,318,336;7,274,316; 7,267,798; 7,260,495; 7,244,570; 7,241,883; 7,234,853;7,230,092; 7,226,737; 7,189,516; 7,141,431; 7,069,191; 7,047,138;6,939,720; 6,916,661; 6,905,766; 6,773,812; 6,696,304; 6,696,265;6,658,357; 6,649,414; 6,632,526; 6,599,331; 6,592,822; 6,528,165;6,524,793; 6,514,295; 6,449,562; 6,411,904; 6,366,354; 6,268,222;6,139,800; 6,057,107; 6,046,807; 5,981,180; 5,802,327 and 5,736,330.

The present invention also includes methods of analyzing and storing thediagnostic data of the present invention. The data stored in thedatabase can be integrated with or compared to other data or databaseswith other SCID markers.

The present invention, therefore, encompasses computer-assisted methodsfor tracking SCID markers encompassing using a computer-based systemcomprising a programmed computer comprising a processor, a data storagesystem, an input device and an output device, and comprising the stepsof generating a SCID profile by inputting into the programmed computerthrough the input device diagnostic data, inputting into the programmedcomputer through the input device diagnostic data, correlating theinputted data with a profile using the processor and the data storagesystem, and outputting a profile to the output device.

The databases and the analysis thereof will be accessible to those towhom access has been provided. Access can be provided through rights toaccess or by subscription to specific portions of the data. The data canbe provided in any form such as by accessing a website, fax, email,mailed correspondence, automated telephone, or other methods forcommunication. These data can also be encoded on a portable storagedevice.

The present invention comprises systems for performing the methodsdisclosed herein. Such systems comprise devices, such as computers,internet connections, servers, and storage devices for data. The presentinvention also provides for a method of transmitting data comprisingtransmission of information from such methods herein discussed or stepsthereof, e.g., via telecommunication, telephone, video conference, masscommunication, e.g., presentation such as a computer presentation (e.g.,POWERPOINT), internet, email, documentary communication such as computerprograms (e.g., WORD) and the like.

Systems of the present invention may comprise a data collection module,which includes a data collector to collect and transmit the data to adata analysis module, a network interface for receiving data from thedata analysis module, and optionally further adapted to combine multipledata from one or more individual patients, and to transmit the data viaa network to other sites, or to a storage device.

More particularly, systems of the present invention comprise a datacollection module, a data analysis module, a network interface forreceiving data from the data analysis module, and optionally furtheradapted to combine multiple data from one or more individual patients,and to transmit the data via a network to other sites, and/or a storagedevice. For example, the data collected by the data collection moduleleads to a determination of the absence or presence of a marker in apatient.

The invention further comprehends methods of doing business by providingaccess to such computer readable media and/or computer systems and/ordata collected from patients to users; e.g., the media and/or sequencedata can be accessible to a user, for instance on a subscription basis,via the Internet or a global communication/computer network; or, thecomputer system can be available to a user, on a subscription basis.

The invention will now be further described by way of the followingnon-limiting examples.

EXAMPLES Example 1 Novel CD3 Epsilon (CD3ε) Peptides

The target protein chosen for these studies is human CD3 epsilon (CDR).It is very similar in the mammalian species that Applicants routinelyuse to produce antibodies (rabbit, mouse). Because of the highsimilarity between sequences it is unlikely that the animal would make adecent immunological response to immunizing peptides and would notproduce high affinity IgG antibody. The sequence for chicken and humanCD3 epsilon are known and have sufficient differences that allowedApplicants to determine 2 potential peptide sequences which would elicitnot only an immune response but would also allow for the development ofhigh affinity IgY:

(SEQ ID NO: 1) 1) AKAKPVTRGAGA (12AA) (SEQ ID NO: 2) 2)LYSGLNQRRI (10AA)

Biologically, IgG antibodies have 3 binding regions. Two regions areused to bind antigen (foreign debris or in this case CD3ε) and the thirdis used to bind different internal receptors for host biologicalfunctions. This can cause a problem with high background when usingantibodies from other mammals to try and detect proteins from humans.This is not the case with chicken antibodies. Chicken antibodiesfunction the same as all the other IgG's, however, the third region onthe IgY is not active in humans, therefore background will be much lowercompared to antibodies produced in other animals.

Laying hens are highly cost-effective as producers of antibodiescompared with other mammals traditionally used for such production. Alsochicken antibodies have biochemical advantages over mammalian antibodiesdue to the phylogenetical differences between avian and mammalianspecies, resulting in increased sensitivity as well as decreasedbackground in immunological assays. In contrast to mammalian antibodies,chicken antibodies do not activate the human complement system nor willthey react with rheumatoid factors, human anti-mouse IgG antibodies, orbacterial and human Fc receptors. Thus chicken antibodies offer manyadvantages over mammalian antibodies and are, even now, beginning toreplace conventional sources of custom produced antibodies (see, e.g.,http://www.oramune.com/custom.aspx?id=7).

Example 2 A Multiplex Immunoassay for SCID Using Guthrie Specimens

SCID (severe combined immunodeficiency) fulfills the requirements for anewborn screening (NBS) condition: sufficient prevalence (estimated at1:50000 to 1:100000); biomarkers in the Guthrie specimen; and theavailability of an appropriate therapy (bone marrow transplant). Anassay to identify SCID and other T-cell deficiencies throughquantification of T-cell recombinant excision circles (TRECs) hasalready been developed and is currently being used in pilot studies inWisconsin and Massachusetts. In those two states, nearly 200,000newborns have been screened without a SCID baby having been found, butother T-cell immunodeficiencies were identified in the screening.Because the TREC assay utilizes DNA technology, those laboratories withlittle experience, equipment or personnel in molecular biology may facechallenges not encountered with an immunoassay. Applicants describe herean alternative test: a multiplex immunoassay that uses the Guthriespecimen, with CD3 as a marker for T-cells, and with CD45 as a markerfor total lymphocytes.

Building on methods and techniques previously described by Applicants,Applicants have developed a two-plex assay that utilizes themultiplexing capability of the Luminex platform. To measure CD3 andCD45, Applicants developed an immunoassay that uses antibody pairs toboth biomarkers. The assays for the two biomarker were developedseparately, and then combined and optimized.

Calibrators were prepared to generate standard curves for CD3 and CD45.These standard curves demonstrate the excellent performance parametersof the antibodies and buffer. The multiplex assay was validated againsteight coded specimens previously characterized by the New EnglandNewborn Screening Program (NESP) laboratory for TREC quantity; five werefrom known SCID cases, and three were controls. All were correctlyidentified by Applicants' new CD345 assay.

Applicants' multiplex immunoassay, using CD3 and CD45 as biomarkers,shows concordance with the TREC assay in detecting immunodeficiencyusing the Guthrie specimen. Its performance characteristics—sensitivity,specificity, and low coefficient of variation are characteristic of animmunoassay. The assay holds promise as an alternative or complement tothe TREC assay for detecting immunodeficies such as SCID. Inclusion inpopulation-based pilot evaluations of SCID NBS is warranted.

SCID (severe combined immunodeficiency) presents one of the greatestopportunities for newborn screening (NBS), and also one of its mostdifficult challenges. This is the first condition detectable by NBS forwhich a cure is available, when identified and treated in early infancy(Railey et al., J Peds 2009; 155(6):834-40). However, the only testcurrently available with the potential of detecting SCID in the Guthriespecimen is the TREC (T-cell recombinant excision circle) assay (Chan &Puck, J Allergy Clin Immunol 2005; 115:391-8). The TREC assay presentstechnical challenges in that it is a primary screening assay using DNA,a protocol not accepted universally by the screening community (Green &Pass, Nat Rev Genet 2005; 6:147-15).

Alternatively, immunoassays are used routinely in NBS as a first-tierscreening protocol (Moyer et al., Hastings Cent Rep 2008; 38(3):32-39).

Applicants report here the technical feasibility of NBS for SCID byimmunoassay, and the development and validation of a multiplex assaythat simultaneously quantifies T-cells and total lymphocytes, in a 3-mmpunch from a Guthrie specimen. The absence of T-cells is the definingcharacteristic of SCID and of other T-cell immunodeficiencies. (Edgar, JClin Pathol 2008; 61:988-93) Because CD3 is part of the T-cell receptorcomplex on mature T-cells, it can be used as a marker for deficiency ofT-cells (Dave, Immunol Rev. 2009; 232:22-33). Two case reports of CD3deficiency causing immunodeficiency have been reported (Roberts et al.,Blood 2007; 109:3198-3206, Rieux-Laucat et al., N Engl J Med. 206;354:1913-21). CD45, a common antigen present on all differentiatedlymphocytes, provides an internal control for the assay. (Eley, CurrAllergy and Clin Immuna March 2008; 21:1-24) Using two different Luminexmicrosphere sets, Applicants developed separate assays for CD3 and CD45,and then combined these and optimized the duplex assay. Anti-CD3specific monoclonal antibody (USBiological) was coupled to Luminex xMAPmicrospheres according to the instructions provided by Luminex(http//Luminexcorp.custhelp.com). Using techniques previously described(10), CD3 capture monoclonal antibody (25-100 μg) was coupled to 5×10⁶Luminex beads, region 32 (L-100-C132-04). Similarly, 25-100 μg of CD45capture monoclonal (USBiological) was coupled to 5×10⁶ Luminex beads,region 33 (L-100-C133-04). Equal volumes of the two coupled bead setswere mixed.

The anti-CD3 polyclonal and anti-CD45 monoclonal detector antibodies(USBiological) were biotinylated with a 20-fold molar excess ofsulfo-NHS-LC-biotin (Pierce). The CD3 detection antibody was used at a1:300 dilution, and the CD45 detection antibody was used at 2 μg/mL.Both capture and detector antibodies to CD45 were selected so that theyrecognized all CD45 isoforms. The performance of the antibodies wasdetermined by titer studies that evaluated affinity and sensitivity, andby cross-reaction tests that evaluated their specificity. Theconcentration of each antibody was titrated so as to produce optimalperformance. The two immunoassays were developed separately andoptimized for affinity, sensitivity, and specificity; they were thencombined into a duplex format.

T-cells and total lymphocytes were counted in whole blood (TennesseeBlood) by flow cytometry; leukocyte-reduced blood (Tennessee Blood) wasexamined by flow cytometry to confirm absence of CD3 and CD45. Theleukocyte-reduced blood was washed four times with PBS, and theresulting packed red blood cells were stored at −80° C. Leukocytes werecollected from the whole blood by use of Histopaque 1077 (Sigma)according to the manufacturer's instructions, counted on ahemacytometer, and resuspended at a concentration of 30×10⁶ cells/mL inhuman serum (BioResource Technology) containing 2% protease inhibitorcocktail (Sigma-Aldrich). Doubling dilutions were carried out to achievea final leukocyte concentration of 0.25×10⁶/mL. An equal volume ofpacked red blood cells was added to each dilution. Leukocyte enrichedblood (75 μL) was spotted on Ahlstrom Grade 226 specimen collectionpaper and left to dry overnight. Dried spots were wrapped in foil andstored in a sealable bag with desiccant at −20° C. Controls were madefrom whole adult blood, with lymphocytes previously measured by flowcytometry.

The assay buffer was prepared using PBS (pH 7.4), 0.055% Tween 20, 0.05%sodium azide, and 0.2% gelatin. To make the elution buffer, 1% proteaseinhibitor cocktail (Sigma) and Triton-X 114 (0.1%) were added to theassay buffer. A single 3-mm punch from a standard, control, or Guthriespecimen was placed in an individual well of a microtiter plate andeluted overnight at room temperature in 100 μL of elution buffer withgentle shaking For the assay, 75 μL of the specimen eluate were added to50 μL of the CD3 and CD45 bead mix, to yield 3000 microspheres per well.After 3 hr incubation with the capture antibodies at 37° C. and gentleshaking, microspheres were washed three times in 100 μL of assay buffer.Next, 50 μL of the anti-CD3 and anti-CD45 detector antibody mixture wereadded to each well. The microplates were incubated for 1 hr at 37° C.with gentle shaking, and the microspheres were again washed three timeswith 100 μL of assay buffer. For detection signal, 50 μL of streptavidinphycoerythrin (Prozyme) was added at 4 μg/mL and incubated for 20minutes at 37° C. The microspheres were aspirated, washed three times in100 μL of assay buffer, re-suspended in 50 μL of 0.2 μg/mLanti-phycoerythrin (Biolegend), and incubated for 30 minutes at 37° C.with gentle shaking. Microspheres were washed three times in 100 μL ofassay buffer, resuspended in 50 μL of 4 μg/mL streptavidinphycoerythrin, and incubated at 37° C. for 20 minutes with gentleshaking Microspheres were aspirated, washed three times with 100 μL ofassay buffer, and then resuspended in 110 μL of Luminex sheath fluid,for analysis.

Data collection and analysis were performed in multiplex acquisitionmode on the Luminex 100 instrument. Luminex software (LX100 IS 2.3)calculated the results, expressed as median fluorescence intensity (MFI)of 100 microspheres of each set. The software LiquiChip Analyser 1.0(Qiagen) was used to analyze the raw data. All control Guthrie specimenswere provided by the New York State Department of Health NewbornScreening Program. In compliance with IRB (Institutional Review Board)guidance, no identifying information was transferred with the specimens.

Calibration curves for CD3 and CD45 quantification in dry blood spots(DBS) are shown in FIG. 2. The analytical limit of detection wasdetermined using the mean plus 3 SD, from 12 replicates of the zerocalibrator. The analytical detection limit in DBS samples for CD3 was0.25×10⁶ cells/mL, and it was 0.125×10⁶ cells/mL for CD45. Accuracy ofthe immunoassay was determined with DBS sample controls made from bloodin which lymphocytes had previously been quantified by flow cytometry.No crossreactivity was observed between CD3 and CD45 antibodies in theduplex format. Using the mean of 12 independent measurements for eachconcentration of calibrators, Applicants examined the assay precisionprofiles. Applicants found that the CVs were approximately 10% for thelower concentrations of CD3, and 12% for the lower concentrations ofCD45. At all concentrations, the inter-assay CV was 3% for CD3, and 1%for CD45. The intraassay CVs for CD3 ranged from 3% to 11% and for CD45from 1% to 12%.

Guthrie specimens from randomly chosen newborns, normal-weight newborns(≧2000 g), and low-birth weight newborns (>500 g-2000 g) were tested todetermine a range for CD3 and CD45. A total of 49 Guthrie specimens weretested: 29 random newborns, 10 normal-birth weight newborns, and 10low-birth weight newborns. To determine T-cell counts, Applicants testedNBS specimens in duplicate in three separate experiments, using thecalibrators described above. Counts ranged from 1 to 13×10⁶/mL cells CD3and from 2.1 to 34×10⁶/mL cells CD45. Specimens from low-birth weightnewborns showed a range from 1.5 to 9.1×10⁶/mL cells for CD3, and from0.5 to 9.7×10⁶/mL cells for CD45 (FIGS. 3 and 4).

The coded punches from New England Newborn Screening Program were testedas singlicates, due to the scarcity of these positive specimens. A setof duplicate random Guthrie specimens was added to the same plate. Areading of “Low” by the software indicates that the value of the markeris too low to compute; likewise a report of “High” denotes that thevalue is above the highest standard. The eight coded specimens wereclassified as positive or negative by the CD345 assay and these resultswere decoded (FIG. 4). Five of the eight 3-mm punches had testedLow/Absent for TRECs, and the remaining three (including one from anadult) were controls. All results from the CD345 assay were concordantwith results from the TREC assay (FIG. 4).

The medical and scientific literature is replete with reports ofsuccessful cures for SCID (Railey et al., J Peds 2009; 155(6):834-40,Griffith et al., J Allergy Clin Immunol. 2008; 122(6):1087-96.Finlayson, Curr allergy and Clin Immunol 2008:21:19-24, Buckley, JAllergy Clin Immunol 2004; 113(4):793-800), thus making it a primecandidate for addition to NBS panels. Currently, the DNA-based TRECassay is the only assay that has been shown to detect lymphopenia in theGuthrie specimen (Puck, J Allergy Clin Immunol. 2007; 120:760-8). Here,Applicants report an alternative, an immunoassay using two T-cellmarkers that was able to differentiate immunodeficient newborns fromcontrols.

This is the first description of the use of CD3 as a biomarker forlymphopenia in an immunoassay of Guthrie specimens, although twoCD3-deficient cases have been documented in the literature (Roberts etal., Blood 2007; 109:3198-3206, Routes et al., JAMA 2009; 302:2465-70).The CD45 biomarker provides an internal control for assay performanceand for the actual presence of a punched sample in each well. It isimportant to note that the values for CD3 in control and affectedinfants differed by 10-fold, thereby assuring adequate separationbetween these two values for other lymphopenias that might be identifiedas assay validation continues. Given the limited number of positivespecimens that were available for evaluation, it is impossible at thistime to establish a cutoff for CD3. As discussed recently, other T-cellimmunodeficiencies can be detected by screening with TRECs (Routes etal., JAMA 2009; 302:2465-70). Applicants believe that the CD345immunoassay described here has comparable potential once it hasundergone further evaluation with positive specimens.

Applicants' CD345 multiplex assay demonstrated concordance of a TRECanalysis on Guthrie specimens, and thus holds comparable potential as acomplement, or substitute, for the TREC assay. Importantly, itsmultiplex format will allow the incorporation of additional markers forSCID into the panel in the future, to improve the specificity, andperhaps also to classify other immunodeficiencies. Additionally,analysis of the punches used for CD345 elution assay (ghosts) in theTREC assay provided concordant results with her first TREC testing. Thisreusable quality of the CD345 test materials validates their use as aprimary screen followed by the TREC as a confirmatory test.

These preliminary data demonstrate that the clinical validity of theCD3-45 multiplex immunoassay is similar to that achievable with the TRECassay. The performance characteristics warrant the assay's inclusion inpopulation-based evaluations.

Example 3 Results of CD3 Epsilon (CD3ε) Peptide Variants Produced bySystematic Single Residue Sequential Replacement Tested in CD3ε ELISA

The capture antibody of the CDR assay was produce by inoculation with 2peptides. The sequence of these two peptides is AKAKPVTRGAGA (SEQ IDNO: 1) and LYSGLNQRRI (SEQ ID NO: 2) respectively. To determine the keyimmune-dominate region of the peptide, 12 variants of original peptide 1(SEQ ID NO: 1) and 10 variants of original peptide 2 (SEQ ID NO: 2) weremade with alanine substitution (or valine when alanine is the originalamino acid). These were produced by GenScript, shipped, in solid form,at room temperature, to our lab and were dissolved in 1 ml of 0.05 TBSbuffer.

Single peptides were coated on to Costar high binding 96 well plates ata concentration of 10 μg and 2 μg in 0.05 TBS buffer pH 7.4 inquadruplicate and tested in an alkaline phoshotase based ELISA. Plateblanks were subtracted from the optical densities obtained for eachpeptide. The peptides are compared to the original by percent oforiginal signal. As noted in Table 1 the twelfth substitution was bothan alanine at position twelve and a glutamic acid for threonine atposition seven. This was an inadvertent oversight by the manufacturer.

The results of these experiments are demonstrated in FIGS. 5A and 5B.The substitution of alanine for residues 8-12 results in 38 to 63%reduction in signal vs. the original peptide 1 (SEQ ID NO: 1) and forresidues 6-10 in original peptide 2 (SEQ ID NO: 2) displayed a 22 to 81%reduction in signal for plates coated at 10 μg/ml. The results fromusing a coating concentration of 2 μg/ml were similar. These experimentsshow that a small change in the peptide sequence will greatly reduce theaffinity with which the anti-human CD38 detection antibody can bind withits recognized epitope.

The OD for original peptide 1 (SEQ ID NO: 1) was 1.5 and peptide 2 (SEQID NO: 2) elicited a signal of 1.0 when tested under the sameconditions. This trend was observed in the three separate experimentsand the conclusion reached is that original peptide 1 (SEQ ID NO: 1)generated an antibody with a greater affinity for peptide 1 than theantibody against peptide 2 (SEQ ID NO: 2).

Example 4 An Improved Immunoassay for the Detection of Severe CombinedImmunodeficiency in Newborn Dry Blood Spots

Applicants previously reported a bead array based multiplex immunoassayfor the detection of human CD3 and all isoforms of human CD45 fromGuthrie specimens. Applicants demonstrated that CD3 could be used as anindication of T cell deficiencies, such as severe combinedimmunodeficiency (SCID), in a 3 mm punch of new born dry blood. HereApplicants report changes to the assay that greatly improve itsperformance and extend its validation.

An antibody pair was used to capture and detect CD3. Applicants replacedthe previously used commercial detection antibody with a custom designeddetection antibody. Assay performance was evaluated and compared withprevious results. Validation of the assay was done with one hundredtwenty four coded 3 mm dry blood specimens obtained from the DanishNewborn Screening Biobank.

The population of coded specimens contained eleven T cell deficientspecimens; all were correctly identified by the CD3 assay. Maternalengraftment (25%) was confirmed in one specimen that was classified asSCID and correctly identified as T cell deficient. Therefore, Applicantshave significantly improved the performance of the CD3 assay.

Testing for SCID has recently become of interest to the newbornscreening (NBS) community, because the detection of this condition inearly infancy is crucial for effective treatment. Several newbornscreening programs have implemented screening for this condition. Thecurrent method of screening for SCID is by T-cell receptor excisioncircles (TREC) assay (Chan K, and Puck J M. J Allergy Clin Immunol 2005;115:391-8; Baker M W et al. J Allergy Clin Immunol 2009; 124:522-7 andGerstel-Thompson J L et al. ClinChem 2010; 56:1466-74), which detectsthe presence or absence of an excised segment of DNA. However, moleculartechniques are not routinely adapted in many NBS laboratories, where animmunoassay would be preferred. Thus, as recently reported (Janik D K etal. J Clin Chem 2010:56(9):1460-69), Applicants developed an immunoassaythat provides an alternative method to TREC testing that quantifies CD3.The rational of Applicants' choice for CD3 is that it is part of the Tcell receptor complex on mature T cells; therefore CD3 can be used as amarker for the presence or absence of T cells (Dave V P. Immunol Rev2009; 232:22-33). Low or absent T cells in peripheral blood is a commoncharacteristic of T cell immunodeficiencies and all but one form ofsevere combined immunodeficiency (SCID) (Edgar J D M. J Clin Pathol2008; 61:988-93). In Applicants' previous report Applicants had a verylimited number of positive SCID specimens for validation. Here,Applicants report the validation of an improved immunoassay in whichApplicants use a custom designed avian anti-human CD3 antibody thatshows superior performance to the previously used commercial antibody onspecimens from SCID affected patients obtained from The Danish NewbornScreening Biobank (Norgaard-Pedersen B et al. J Inherit Metab Dis. 2007;30: 530-6).

Specimens were retrieved from The Danish Newborn Screening Biobank. Thestudy was accepted by The Danish Data protection Agency(no:2010-41-4335), by The Steering Committee for the Newborn ScreeningBiobank and the New York State Department of Health's IRB 00-402. Allspecimens were coded and the diagnosis was not known to us until testingwas completed.

The anti-human CD3 capture antibody was purchased from USbiological. TheIgY anti-human CD3 detection antibody was made in chicken viaInvitrogen's (Carlsbad, Calif.) custom antibody service and delivered ata stock concentration of 1 mg/mL. Other reagents used were:Sulfo-NHS-LC-biotin (Pierce, Rockford, Ill.);streptavidin−Phycoerytherin (Prozyme, Hayward, Calif.); phosphatebuffered saline+Tween 20, protease inhibitor cocktail, gelatin, andHistopaque 1077 (Sigma, St. Louis, Ill.); whole and leuko-depleted bloodunits (Tennessee Blood Services, Memphis, Tenn.); carboxylated xMAPmicrospheres (Luminex Corp., Austin, Tex.); low protein binding 96-wellfilter bottom plates (Millipore, Billerica, Mass.), flat bottommicrotiter plates (Corning, Corning, N.Y.); pooled human serum(BioResource Technology, East Greenwich, R.I.); triton-x114 (MPBioscience, Solon, Ohio), and Ahlstrom grade 226 specimen collectionpaper (ID Biological Systems, Greenville, S.C.)

Anti-CD3 specific capture monoclonal antibodies were coupled to LuminexxMAP microspheres following the protocol provided by Luminex(http://www.luminexcorp.com/support/protocols/index.html). Usingtechniques previously described (Bellisario R et al. Clin Chem 2000;46:1422-4; Bellisario R et al. Early Hum Dev 2001; 64:21-25 andLindau-Shepard B A and Pass K A. Clin Chem. 2010. 56:3:445-450), 25-100μg of anti-CD3 capture monoclonal antibody was coupled to 5×10⁶ Luminexmicrospheres, region 132 (L-100-C132-04). The anti-CD3 polyclonalantibody was biotinylated with sulfo-NHS-LC-biotin according to themanufacturer's instructions (Pierce, Rockford, Ill.).

Whole blood was used to prepare calibrators and controls, afterdetermining leukocyte counts by flow cytometry. Leukocytes werecollected from whole blood by use of Histopaque 1077, according to themanufacturer's instructions, counted on a hemacytometer, and resuspendedat a concentration of 30×10⁶ cells/mL in human serum containing 2%protease inhibitor cocktail. To make standards, two-fold serialdilutions of the 30×10⁶ cells/mL suspension were carried out in humanserum containing protease inhibitors to achieve a final leukocyteconcentration of 0.234×10⁶ cells/mL.

Leukocyte-reduced blood was examined by flow cytometry to confirm theabsence of CD3 and CD45 positive cells. Packed red blood cells were madeby washing leukocyte-reduced blood four times with phosphate bufferedsaline (PBS), pH 7.4 with centrifugation at 3000 g for 30 min. An equalvolume of packed red blood cells (RBC) was added to each dilution toachieve a hematocrit of 50%. The leukocyte-enriched blood (75 μL) wasspotted on Ahlstrom Grade 226 specimen collection paper and driedovernight. Dried spots were wrapped in foil and stored in a sealable bagwith desiccant at −20° C. Controls were made from whole adult blood,with lymphocytes previously measured by flow cytometry.

Enhancement to the protocol from the previously published assay (Janik DK et al. J Clin Chem 2010:56(9):1460-69) included replacing thecommercially available anti-CD3 detection antibody with a custom madeIgY anti-CD3 detection antibody used at a concentration of 0.25 mg/L,and the incubation time of blood eluate and microspheres was reducedfrom 3 hours to 2 hours and the amplification step and its associatedwash steps have been removed. The enhanced assay protocol consisted ofsix steps with washings between each step; incubation of eluate andmicrospheres, wash, incubation with detection antibody, wash, incubationwith streptavidin phycoerythrin, wash, resuspend and read.

Standard curve comparison of the commercial CD3 detection antibody tothe IgY detection antibody showed a broader linear detection range forthe IgY antibody. The chicken antibody required less incubation time andrequired fewer reagents per sample. The IgY detection antibody had alinear range from 0.2×10⁶ to 15×10⁶ T cells/mL, while the commercialantibody had a linear range of 1.0×10⁶ to 7.5×10⁶ T cells/mL (FIG. 6).

Applicants measured the CD3 concentration of 124 coded neonate dry bloodspots obtained from The Danish Newborn Screening Biobank. Meanfluorescent intensities were converted to concentrations of CD3 usingthe LiquiChip program.

The decoded results revealed that normal infant samples had T cellcounts which ranged from 2.14×10⁶/mL to 27.8×10⁶/mL, while samples fromaffected infants ranged from 0.03×10⁶/mL to 1.07×10⁶/mL (FIG. 7). Elevensamples were from infants with T cell related immunodeficiencies (FIG.8); eight of the eleven samples were a form of SCID, one sample was SCIDwith 25% maternal engraftment and the remaining two specimens werediagnosed with Omenn syndrome and Wiskott Aldrich syndrome. Four controlsamples were labeled as High and 5 of the affected samples were labeledas Low. A value of High or Low by the Liquichip software indicates thatthe measured MFI values were too high or too low to extrapolate aconcentration.

In Applicants' previously described immunoassay (Janik D K et al. J ClinChem 2010:56(9):1460-69) Applicants utilized the most effectivedetection antibody that was commercially available. However, because oflot to lot variation and availability issues Applicants felt a morereliable supply of antibody was necessary to make a dependable assay.Applicants contracted for the production of antigen specific avian(chicken) antibodies (IgY) to human CD3 because IgY antibodies have beenshown to be stable for long term storage and do not bind to human Fcreceptors, rheumatoid factor or complement, thus, nonspecific binding ofinterfering substances is greatly reduced. IgY antibodies are purifiedfrom chicken egg yolks with an egg laying hen producing enough eggs in aweek to purify antibodies equivalent to the amount of antibodies in75-90 mL of mammalian serum (Larsson A. et al. Poultry Science. 1993.72(10):1807-12). Indeed, Applicants' study demonstrates that the customIgY antibodies to CD3 have low background and are highly specific.

To date, the CD3 immunoassay Applicants developed using these antibodieshas been able to correctly identify specimens with low or undetectable Tcells from various SCID forms, Wiskott-Aldrich Syndrome, Ommen Syndromeand two SCID specimens with maternal engraftment. With the enhancedperformance and ability of the CD3 assay to correctly identify T celldeficiency, it should be considered a complimentary or alternative toother dry blood spot T cell detection techniques.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

1. An isolated CD3 epsilon peptide comprising residues 8-12 of SEQ IDNO:
 1. 2. An isolated CD3 epsilon peptide comprising residues 6-10 ofSEQ ID NO:
 2. 3. (canceled)
 4. An isolated CD3 epsilon peptideconsisting of 10 residues wherein residues 6-10 of the peptide areresidues 6-10 of SEQ ID NO:
 2. 5. (canceled)
 6. An isolated CD3 epsilonpeptide consisting of SEQ ID NO:
 2. 7. An isolated antibody against thepeptide of claim
 1. 8. An isolated antibody against the peptide of claim2.
 9. An isolated antibody against the peptide of claim
 3. 10. Anisolated antibody against the peptide of claim
 4. 11. An isolatedantibody against the peptide of claim
 5. 12. An isolated antibodyagainst the peptide of claim
 6. 13. A method of identifying animmunodeficiency in a patient, comprising isolating a blood sample fromthe patient and performing an immunoassay on the blood sample with theantibody of any one of claims 7-12.
 14. The method of claim 13, whereinthe immunodeficiency is severe combined immunodeficiency (SCID).
 16. Themethod of claim 13, wherein the immunodeficiency is a T cellimmunodeficiency.